Two single strands of deoxyribo- ("DNA") or ribo-("RNA") nucleic acid, formed from nucleotides, (including the bases adenine (A), cytosine (C), thymidine (T), guanine (G), uracil (U), or inosine (I)), may hybridize to form a double-stranded structure held together by hydrogen bonds between pairs of complementary bases. Generally, A is hydrogen bonded to T or U, while G or I are hydrogen bonded to C. Along the chain, classical base pairs AT or AU, TA or UA, GC, or CG are present. Additionally, some mismatched base pairs (e.g., AG, GU) may be present.
Bringing together two single strands of nucleic acid containing sufficient contiguous complementary bases, under conditions which will promote their hybridization, results in double-stranded nucleic acid. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids can form.
A probe is generally a single-stranded nucleic acid sequence complementary to some degree to a nucleic acid sequence sought to be detected ("target sequence"). A probe may be labeled with a reporter group moiety such as a radioisotope, a fluorescent or chemiluminescent moiety, or with an enzyme or other ligand which can be used for detection. Background descriptions of the use of nucleic acid hybridization to detect particular nucleic acid sequences are given in Kohne, U.S. Pat. No. 4,851,330 issued Jul. 25, 1989, and by Hogan et al., International Patent Application No. PCT/US87/03009, entitled "Nucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organisms," both references hereby incorporated by reference herein. Hogan et al., supra, describe methods for determining the presence of a non-viral organism or a group of non-viral organisms in a sample (e.g., sputum, urine, blood and tissue sections, food, soil and water).
The genera Ureaplasma and Mycoplasma are prokaryotes and comprise the taxonomic Mollicutes class. Mollicutes lack a bacterial cell wall and have a small genome size. They are considered one of the smallest of the free-living microorganisms. Ureaplasma are unique among Mollicutes because of their characteristic ability to metabolize urea. There are fourteen known serotypes of U. urealyticum (Stemke and Robertson, Diagn. Microbiol. Infect. Dis. 31: 311 (1985)). The fourteen serotypes can be divided into at least two subspecies ("biotypes") based upon restriction fragment length polymorphism ("RFLP") of U. urealyticum genomic DNA (Harasawa et al., Abstract S30-6 UIMS Meeting, Osaka Japan (1990), and Robertson et al., J. Clin. Microbiol. 31: 824 (1993)), or based upon rRNA sequences (Hammond et al., Abstract D17. Session 60, American Society for Microbiology General Meeting, (1991)).
U. urealyticum is commonly found in the human urogenital tract but has been implicated in a wide spectrum of pathologies. Several studies have implicated U. urealyticum as a possible etiologic agent in diseases affecting adult males, fetuses and infants. Brunner et al., Yale J. Biol. Med. 56: 545 (1983), identified U. urealyticum as the etiologic agent responsible for nongonococcal urethritis (NGU) in approximately 30 percent of adult males tested who had NGU. Cassell et al., Pediatr. Infect. Dis. 5: S247 (1986), implicated U. urealyticum as a possible cause of chorioamnionitis, which could in turn adversely affect the outcome of pregnancy and the health of neonates. Stagno et al., Pediatrics 68: 322 (1981), found U. urealyticum in 21% of infants with pneumonia and found the association of U. urealyticum with pneumonia to be "statistically significant." Waites et al., Lancet 8575: 17 (1988), found U. urealyticum in 8 percent of the cerebrospinal fluid specimens taken from a high-risk population of newborn infants (100 predominantly pre-term infants). According to these investigators U. urealyticum was the most common organism isolated of those sought. U. urealyticum has also been implicated in a number of other pathogenic states including septic arthritis (Lee et al., Arthritis and Rheumatism 35: 43 (1992)).
Standard microbiological techniques generally identify U. urealyticum by observing the hydrolysis of urea. These techniques usually involve inoculating both a complex broth medium and an agar medium containing urea and other nutrients with a freshly obtained specimen (Brunner et al., supra).
References concerning detection of Ureaplasma include the following: Roberts et al., Israel J. Med. Sci., 23: 618 (1987), describe the use of whole chromosomal DNA probes for detection of Ureaplasma in genital specimens; Ohse and Gobel, Israel J. Med. Sci. 23: 352 (1987) describe hybridization of U. urealyticum rRNA genes to cloned DNA of the E. coli rRNA operon; Gobel and Stanbridge ("Detection of Mycoplasma by DNA Hybridization", EPO application number 86304919.3, publication number 0 250 662) mention biological probes for detecting Mycoplasmas or prokaryotes in general, or specific Mycoplasma and eubacterial species; Gonzales et al. (American Society for Microbiology Annual Meeting 1991, Abstract D-16) mentions a method to detect Ureaplasma using a DNA probe directed to rRNA; Lee et al., supra, and Willoughby et al., Infection and Immunity 59: 2463 (1991), describe a procedure for detecting the U. urealyticum urease gene utilizing PCR; Deng et al., PCR Methods and Applications 1: 202 (1992), suggest that PCR-RFLP techniques should be capable of detecting Mollicutes; Brogan et al., Molecular and Cellular Probes 6: 411 (1992), describe the amplification of a 186 base pair genomic U. urealyticum DNA fragment; Robertson et al., supra, describe a technique involving the polymerase chain reaction using biotype specific primers to 16S rRNA gene sequences to distinguish the two U. urealyticum biotypes.